Altered vertebrae morphology and bone mineralization in a ...Jul 10, 2020 · 3 56 Introduction 57...

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Altered vertebrae morphology and bone mineralization in a zebrafish model of CHARGE 1

syndrome 2

Maximilian Breuer1*; Maximilian Rummler2*; Charlotte Zaouter1; Bettina M. Willie2#; 3 Shunmoogum A. Patten1,3# 4

*, # authors contributed equally 5

Affiliations: 6

1. INRS – Centre Armand Frappier Santé Biotechnologie, 531 Boulevard des Prairies, Laval, 7 QC, Canada, H7V 1B7 8 2. Research Centre, Shriners Hospital for Children-Canada, Department of Pediatric Surgery, 9

McGill University, 1003 Decarie Blvd, Montreal, Canada H4A 0A9 10

3. Centre d'Excellence en Recherche sur les Maladies Orphelines - Fondation Courtois 11

(CERMO-FC), Université du Québec à Montréal (UQAM), Montréal, QC , Canada 12

Key words: CHARGE syndrome, microCT, Zebrafish, late onset idiopathic scoliosis, 13 osteogenesis 14

*Correspondence: Correspondence should be addressed to 15 Dr. Maximilian Breuer 16 INRS- Centre Armand-Frappier Santé et Biotechnologie 17

531 Boulevard des Prairies 18 Laval, Quebec 19

H7V 1B7 20 Canada 21

[email protected] 22 +1 (438) 341-0445 23 24

Conflict of Interest 25

The authors declare no conflicts of interest 26

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(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted July 11, 2020. . https://doi.org/10.1101/2020.07.10.197533doi: bioRxiv preprint

mailto:[email protected]

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Abstract 33

CHARGE syndrome patients present features of idiopathic scoliosis in over 60% of cases, 34

reduced bone mineral density and in a few cases osteopenia. While several clinical cases and 35

studies regarding the spinal deformities in CHARGE syndrome bearing CHD7 mutations are 36

well-documented, the underlying mechanisms remain elusive. Here, we detect and 37

quantitatively analyze skeletal abnormalities in adult and young chd7-/- larvae. 38

We show that young chd7-/- larvae present with scoliosis and kyphosis already at 9 dpf. Gene 39

expression analysis confirmed the reduction of osteoblast markers and Pparγ targets. MicroCT 40

analyses identified abnormal Weberian apparatus structure and vertebral body morphology in 41

chd7-/- mutants, with highly mineralized inclusions, along with variances in bone mineral 42

density and bone volume. Additionally, we detect a specific depletion of Col2a1a in the 43

zebrafish vertebral cartilage, in line with a significantly reduced number of chondrocytes. 44

Our study is the first to elucidate the mechanisms underlying morphological changes in 45

vertebrae of adult chd7-/- zebrafish and decreased spinal integrity. The chd7-/- zebrafish will be 46

beneficial in future investigations of the underlying pathways of spinal deformities in CHARGE 47

syndrome. 48

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Introduction 56

Impaired bone development and spinal dysmorphisms are a major health concern in various 57

genetic disorders. Among these, Idiopathic scoliosis is a commonly observed phenotype and 58

the most common skeletal abnormality in children.(1) Idiopathic scoliosis is a curvature of the 59

spine with unidentified genetic cause. However, many risk genes have been connected to the 60

underlying mechanisms.(2) One of these related genetic disorders is the congenital 61

multisystemic CHARGE syndrome (CS) named after the major characteristics of Coloboma, 62

Heart defects, Atresea chonae, Retarded growth, Genital and Ear abnormalities.(3-5) Though, 63

considering the wide variety of observed phenotypes, the list of characteristics has been 64

continuously augmented to include craniofacial abnormalities and spinal deformations.(6-8) 65

Along these lines, CS is closely associated with skeletal deformities such as idiopathic scoliosis, 66

kyphosis and hemivertebrae.(9) In fact, idiopathic scoliosis is observed in a majority of CS 67

cases, with studies showing over 60% of patients having diagnosed scoliosis at an average age 68

of just over 6 years.(10-12) In some cases, the areal bone mineral density (aBMD) is reduced. (13) 69

CS is most commonly caused by a mutation in the chromodomain ATP-dependant helicase 7 70

(CHD7). Mutations are distributed evenly throughout the gene and the vast majority of cases 71

are sporadic, with only very few cases of familial CS.(7, 14-16) Analysis of pathways related to 72

CHD7 show involvement in neural crest differentiation/proliferation/migration and stem cell 73

quiescence.(17-19) Furthermore, studies focusing on CS have identified regulatory mechanisms 74

of CHD7 in the immune response and more strikingly in brain development.(20, 21) Most 75

recently, CHD7 has been connected to abnormal GABAergic development resulting in an 76

autistic-like behavior (Jamadagni et al., unpublished data). While the function of CHD7 has 77

been extensively studied, little attention has been given to the underlying mechanisms in 78

skeletal development.(22) Some studies have directly linked CHD7 to osteogenesis. (23, 24) 79

Investigation in cell cultures have shown a dependency on CHD7 for successful differentiation, 80


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more specifically to a differentiation complex with SETD1B, NLK and SMAD1 resulting in 81

depleted osteogenesis upon depletion of CHD7 in favour of adipogenesis by regulating PPARγ 82

target genes.(23, 24) Various models to investigate this role in vivo have been proposed. A mouse 83

model for Chd7 deficiency, termed “looper” presents with ear ossicle malformations, but no 84

spinal deformities.(25) However, mouse models for Chd7 deficiency have limitations, as null 85

mutants are embryonically lethal by 10 days.(26) Yet, a recent zebrafish model has proven 86

valuable in the modelling of chd7 dependent CS revealing a reduction in vertebrae 87

mineralization of young larvae.(27) 88

Zebrafish have become increasingly relevant in the study of fundamental bone development 89

and bone related disorders(28-30), including scoliosis, osteoporosis, age related osteoarthritis.(31, 90

32) Notably, the spinal structure in zebrafish has been characterized in detail and comprises three 91

major regions: the Weberian apparatus, a specialized structure consisting of the first four 92

vertebrae connecting the auditory system and the swim bladder to amplify sound vibrations, the 93

precaudal vertebrae which are connected to neural arch and spines to form the chest and, the 94

caudal vertebrae of the tail region with neural and hemal arch.(33) Simplicity of analysis in 95

zebrafish to investigate spinal structures has been used to understand the effects of mechanical 96

loading on bone mineralization.(30, 34) Zebrafish have specific advantages in the analysis of 97

skeletal development such as the closely related structure to humans, rapid bone development 98

with first mineralization of vertebrate occurring after only 5 days post fertilization (dpf), as well 99

as ex utero development and transparency of young larvae simplifying the use of in vivo staining 100

and transgenic lines to allow for the analysis of early calcification in zebrafish bony 101

structures.(35-38) Furthermore, the simplicity of high-throughput drug screening in this teleost 102

model makes the model highly useful for investigating new drug targets and effect on skeletal 103

development.(39, 40) These advantages have raised interest due to their high remodelling 104

efficiency of skeletal structures including their possibility to regenerate fins, as well as the 105


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effectiveness to reproduce spinal deformity phenotypes.(41, 42) Some of these studies in zebrafish 106

show a close link to the Collagen Family.(43, 44) These highly conserved pathways between 107

zebrafish and humans allow us to investigate the underlying pathomechanisms linking CHD7 108

to idiopathic scoliosis and other skeletal abnormalities. 109

We recently generated a stable chd7-/- zebrafish mutant using CRISPR/Cas9 that replicates 110

hallmarks of CS (Jamadagni et al., unpublished data), consistent with our previous findings 111

using a transient chd7 morpholino knockdown model.(27) In this study we describe for the first 112

time a detailed analysis of skeletal development in a zebrafish chd7-/- mutant model for CS. 113

Early larvae screens reveal a delay of mineralization of vertebrae bodies linked to deficient 114

osteoblast differentiation. Gene expression and IHC analysis reveals striking reduction in 115

osteoblast, chondrocyte, and collagen matrix markers, particularly displaying diminished levels 116

of Col2a1. Finally, extensive MicroCT analysis in adults show abnormal mineralization and 117

morphological structures in precaudal and caudal vertebrae. 118

Results 119

Zebrafish chd7-/- larvae show spinal deformities 120

To investigate if spinal deformities are present at an early developmental stage, we screened 121

young chd7-/- larvae at 9 dpf (n=110) for morphological abnormalities. 39% of chd7-/- larvae 122

exhibited highly varying, scoliosis-like phenotypes at both precaudal and caudal regions of the 123

spine (Fig. 1a). Additionally, some of these also presented with a kyphosis-like phenotype 124

(Supplemental Fig. 1a). Even with the morphological phenotype, fish were reactive to a touch 125

response. 126

chd7-/- larvae have a spinal mineralization deficit that is nutrient dependent 127

Given the spinal deformities in 9dpf chd7-/- larvae, we next sought to investigate the efficiency 128

of mineralization during larval development. Calcein staining for fluorochrome labeling of 129


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bones revealed no delay in the onset of mineralization of the first precaudal vertebrae at 6 dpf. 130

Upon further development, at 9dpf, chd7-/- larvae showed a significant reduction in the number 131

of calcified vertebrae (Fig. 1c). Notably, chd7-/- larvae were less efficient in complete 132

calcification of vertebrae towards the posterior region of the spine (Fig. 1b, c). 133

Since calcification of the bone matrix is dependent on mineral uptake from food, we tested how 134

the onset and development of vertebral bone structure calcification in 9 dpf chd7-/- zebrafish 135

was dependent on nutrition accessibility. Notably, wildtype and chd7-/- larvae showed no 136

difference in the number of mineralized vertebrae in a starvation situation between 5 dpf and 9 137

dpf. Expectedly, we observed significant differences when food was available from 5 dpf until 138

9 dpf, with chd7-/- zebrafish having a reduced number of mineralized vertebrae compared to 139

controls. The chd7-/- larvae had the same number of mineralized vertebrae when food was 140

available or absent (Fig. 1b, d). Starvation in zebrafish larvae between 5 dpf and 9 dpf also 141

resulted in smaller yolks, which is to be expected, considering the high dependency on nutrition 142

supply by the yolk. Surprisingly, we found that Weberian vertebrae, which are the first 4 143

vertebrae of the spinal column, were more rapidly calcified in chd7-/- larvae than in controls 144

(Fig. 1b arrows, Fig. 1e). 145

We investigated if mineralization was affected in later development. Insufficient mineralization 146

was also evident in 4-week-old juvenile zebrafish with spinal deformities. Alizarin red staining 147

revealed decreased mineralization of caudal vertebrae, with striking deficiency toward the 148

vertebral body (Fig. 1f). 149

Osteoblast differentiation is impaired in chd7-/- larvae 150

CHD7 has been shown to regulate osteogenesis by controlling PPARγ to promote skeletal 151

development and regulate expression of promoting genes. To understand if the Pparγ pathway 152

is directly involved in the observed bone mineralization deficiency and abnormal morphology 153

in chd7-/- larvae, we tested for Pparγ target gene expression and osteoblast differentiation 154


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marker genes at the onset of the defect at 9 dpf (Fig 1g). The key target for Pparγ, runx2a and 155

runx2b, were significantly downregulated in chd7-/- larvae compared to controls. Further 156

markers for osteogenesis were affected, including a significant downregulation of osteoblast 157

markers sp7/osterix, bglap and postna. The chd7-/- larvae also had significantly lower levels of 158

osteocyte markers and other genes regulating mineralization such as acp5a and sost, while mgp 159

remained unchanged compared to control larvae. Additionally, we detected a significant 160

upregulation of osteoclast marker ctsk in chd7-/- larvae. 161

Abnormal development of the Weberian apparatus in adult chd7-/- zebrafish 162

To assess the effects of chd7 deficiency on vertebral growth and maturation, we analyzed 163

vertebrae morphology and mass using microCT in adult zebrafish. Morphologically, adult chd7-164

/- zebrafish were unchanged in body length, however, appeared strikingly leaner in body stature 165

compared to adult control zebrafish. 166

Since mineralization in the young larvae varied from anterior to posterior, we decided to 167

analyze the three major structures of the zebrafish spinal column: The Weberian apparatus, 168

precaudal and caudal vertebrae. The Weberian apparatus and its specialized structures, the 169

supraneurals, intercalarium, tripus and parapophysis, were thinner and smaller in morphology 170

in chd7-/- adults in comparison to controls (Fig. 2 b). We further performed a detailed analysis 171

of volumetric bone mineral density (vBMD), bone volume (BV), total volume (TV), and bone 172

volume fraction (BV/TV) of 1-year-old chd7-/- larvae and control zebrafish. We did not detect 173

any significant differences in vBMD, BV or TV (Fig. 2 c-h). However, we found a significantly 174

greater variance in TV of both intercalarium and parapophysis, as well as in BV of the 175

parapophysis in 1-year-old chd7-/- zebrafish, as determined by F-test analysis (Supplemental 176

Table 1). The same results were observed in 2-year-old chd7-/- compared to control zebrafish 177

(Supplemental Table 2). 178


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Abnormal mineralization and malformations in the body end plates of precaudal vertebrae in 179

adult zebrafish 180

Vertebrae structures were analyzed by assessing key angles of the structure (Fig 3a) and 181

mineralization features. MicroCT analysis of precaudal vertebrae identified abnormal patterns 182

of mineralization and abnormal structures, that were especially pronounced at the arches and 183

transverse processes in 1-year old chd7-/- and control zebrafish (Fig. 3b, c). Precaudal vertebrae 184

showed significantly higher overall vBMD, while the distribution of mineralization between 185

the body and arch remained unaffected (Fig. 3d, e). However, structurally, precaudal vertebrae 186

of chd7-/- mutants show an increase of bone volume towards the body end plates of vertebrae, 187

which in most cases show major malformations manifesting as highly mineralized inclusions 188

(Fig. 3b, arrows). Additionally, vertebrae in 1-year-old chd7-/- zebrafish presented with a larger 189

BV/TV compared to controls (Fig. 3g-i). Analysis of the vertebrae neural arch revealed 190

distortion with a significantly larger rising angle, as well as a larger variance of the angles (Fig. 191

3f). Vertebrae showed a significantly changed body angle and significant variance of body 192

angle and vertebrae length (Supplemental Table 3). While a depletion of vBMD in the arches 193

and abnormal vertebrae body structure was observed in 2-year-old fish, the effects seen on total 194

vBMD and BV were not apparent in the few samples that survived until this stage 195

(Supplemental Fig. 2 and Supplemental Table 4). Notably, one of our chd7-/- mutants showed 196

vertebral fusion of the analyzed precaudal vertebra and following vertebra, which was not 197

observed in any of the controls (Supplemental Fig. 1). This further demonstrates the wide 198

variation in severity of the bone phenotype in chd7-/- zebrafish. 199

Abnormal caudal vertebrae in chd7-/- zebrafish vary in arch angles and reduced mineralization 200

We next determined key structural features (Fig. 4a) and mineralization characteristics of 201

caudal vertebrae. The chd7-/- zebrafish presented with warped hemal and neural arches and 202

abnormal body structure compared to controls (Fig. 4b). Similar to precaudal vertebrae, the 203


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growth zones of the body end plates were enlarged and showed inclusions of highly mineralized 204

matrix (Fig. 4b). Unlike control zebrafish, chd7-/- mutants had abnormal vertebrae body 205

structure, which usually exhibited an hourglass shape and resulting in greater variance of 206

measured body angles (Supplemental Table 3). 207

In line with the neural arch screened in the precaudal vertebrae, the hemal arch in the caudal 208

vertebrae showed significantly wider arch rising angles (Fig. 4f), while the neural arch 209

displayed significantly greater variance (Fig. 4g) in 1-year-old chd7-/- zebrafish. In contrast to 210

precaudal vertebrae data, the caudal vertebrae showed significantly larger variance of Total 211

vBMD and of both arches and vertebral body vBMD (Supplemental Table 3). A significant 212

insufficiency in mineralization of the arches compared to the vertebrae body was also measured 213

in chd7-/- zebrafish compared to controls (Fig 4 d, e). Further analysis also revealed a significant 214

variance in BV, TV and BV/TV in chd7-/- zebrafish compared to controls (Fig h, i). 215

While significantly distorted arches were still observed in 2-year old mutants, the effects on 216

vBMD and BV were not noticed in the few surviving fish that could be screened (Supplemental 217

Fig. 3 and Supplemental Table 4). 218

Col2a1 of the ECM is depleted in chd7-/- zebrafish 219

To obtain a more complete analysis of vertebrae integrity, we analyzed the extracellular matrix 220

(ECM) structures of the vertebral column in 9 dpf larvae and 1-year-old chd7-/- zebrafish and 221

controls. Hematoxylin-Eosin (HE) staining of precaudal vertebrae sections showed a significant 222

reduction in the number of nuclei measured in the vertebral cartilage of 1-year-old chd7-/- 223

zebrafish and controls (Fig. 5a, b). More detailed analysis using Safranin/Fast Green O staining, 224

revealed a significant reduction in the number of chondrocytes in the vertebral cartilage in chd7-225

/- zebrafish in line with the HE staining (Fig. 5c, d). Additionally, both stainings indicate a 226

hypertrophic cell morphology in chd7 mutants. We further screened for major components of 227

the cartilage matrix. Gene expression analysis at 9 dpf revealed a significant downregulation of 228


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the ECM related gene col2a1a (Fig. 5e). Immunofluorescence showed a striking depletion of 229

Col2a1 in the vertebral cartilage with few cells remaining, which produce a sufficient Col2a1 230

signal (Fig 5f). This pattern was effectively reproduced in all regions of the spine of chd7-/- 231

zebrafish. 232

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Discussion 234

Efficiency of model 235

CS is a multisystemic disorder likely involving a variety of underlying mechanisms that include 236

CHD7. Therefore, different preclinical models are required to individually identify the various 237

molecular mechanisms. Even though mouse models for CS exist, none have been reported to 238

show the bone phenotype observed in CS patients. Our study presents a chd7 deficient zebrafish 239

that has a bone phenotype resembling that of CS patients, which we used to investigate skeletal 240

development in CS. While scoliosis in CS patients has been attributed to poor muscular 241

development, our study proposes an additional factor involving impaired early mineralization 242

of vertebrae. In line with observations in patients, we observed spinal deformities in chd7-/- 243

zebrafish, indicative of scoliosis.(10-12) Interestingly, our analysis reveals vertebral abnormalities 244

are already present in young larvae of chd7-/- zebrafish. Additional clinical studies are required 245

to investigate the onset of spinal deformities in CS patients. However, early analysis of bone 246

density and spinal development has been suggested by CS guidelines.(45, 46) Detailed analysis 247

may reveal less severe or early onset phenotypes. Similarly, we observed that adult chd7-/- 248

zebrafish, which appeared without spinal deformities on visual inspection, showed signs of 249

spinal abnormalities in microCT analysis, suggesting that mostly mild phenotypes reach this 250

age. This is supported by the high lethality rate shown by Jamadagni et al. (unpublished), yet 251

the underlying cause of lethality is still unclear. 252


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Progressive skeletal disorders such as osteopenia and osteoporosis are characterized by a 253

reduction in bone mineralization over time leading to increased fracture risk.(47) CS patients 254

have been shown to present with significantly reduced bone mineral density.(13) 255

Correspondingly, reduced vertebrae mineralization in chd7-/- zebrafish by 12 dpf was observed 256

in a previous study involving morpholinos in zebrafish which had observed a reduced 257

mineralization of vertebral structures.(27) Confirming previous data, we observed insufficient 258

mineralization of vertebra along the posterior spinal column by 4 weeks of age. In earlier 259

analysis of larvae at 6dpf, we were not able to detect a delay in the onset of mineralization, but 260

already by 9 dpf we saw a significant decrease in the number of calcified vertebrae. 261

Furthermore, mineralization appeared to be less efficient in posterior vertebrae, which could 262

signify an impairment in ossification, since vertebrae in zebrafish undergo ossification from an 263

anterior to posterior direction.(48) An exception from this rule is the Weberian vertebrae C1 and 264

C2, which are ossified later in wildtypes but earlier in chd7-/- mutants. 265

Nutrient dependency 266

Our study showed that the onset of calcification was not delayed in chd7 deficient zebrafish. 267

However, progressing calcification was decreased already 4 days post feeding onset, even 268

though access to food was identical in control and mutants. In contrast, differences in 269

calcification of vertebrae in control and mutants without access to food was not significant. 270

Previous studies have shown that feeding delay until 9 dpf does not affect future fish growth 271

and viability and was therefore our cut-off point.(49) This data suggests that possibly an 272

underlying mechanism involving either reduced uptake or metabolizing nutrients may play a 273

part in the observed phenotype and this link remains to be investigated. Bone calcification and 274

mineralization is dependent on proper nutrition and is often discussed regarding optimal bone 275

health in humans.(50) Insufficient levels of calcium and nutritional uptake are considered an 276

increased risk for bone injury in CS patients.(11) To optimize nutritional uptake, the 277


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supplementation of calcium and other nutrients to counteract poor bone health has been 278

proposed for CS patients.(51) In fact, case reports show supplementation with calcium and 279

vitamin D significantly increased aBMD in CS patients .(52) A possible nutrient deficiency in 280

CS, could be linked to gastrointestinal nutrient uptake difficulties in chd7-/- zebrafish(53), yet 281

this needs to be further investigated. Thus, this possible deficiency in nutrient uptake could 282

further hinder ossification in chd7-/- mutants. We propose using our zebrafish model of CS 283

model to test the effect of guidelines such as calcium and nutrient supplementation, nutrient 284

metabolism and mechanical loading in form of swim training to enhance bone strength in CS. 285

Loss of chd7 reduces osteogenesis in zebrafish 286

Osteoblast precursors commonly express differentiation markers such as runx2 and 287

Osterix/sp7.(54) Upon maturation of these osteoblasts, expression of mineralization related 288

genes such as bglap (osteocalcin) are expressed. Analysis of these three genes showed that they 289

were all significantly downregulated in chd7-/- larvae. This suggests a deficiency in mature 290

osteoblasts, required for the mineralization of the spinal column. Misregulation or loss of theses 291

genes, such as osterix/sp7 has been directly linked to poor bone mineralization in zebrafish and 292

humans.(55-57) Notably, among all osteogenesis related gene expressions significantly reduced 293

in chd7-/- larvae, sost, ctsk, acp5 and col2a1 have been identified as direct regulatory targets of 294

Chd7 by Chip-Seq.(58) Furthermore, the regulation of osteogenesis has been linked to a 295

regulatory complex with PPARγ.(23) Cell culture experiments have proposed the role of CHD7 296

in osteogenesis by inhibiting PPARγ target genes by a protein complex involving CHD7, NLK 297

and SETDB1. Upon loss of CHD7 this complex will fail to inhibit PPARγ target genes and 298

therefore inhibit osteogenesis in preference over adipogenesis.(23) Strikingly, our qPCR results 299

are consistent with downregulation of Pparγ target genes such as runx2a and runx2b. 300

Furthermore, gene expression relevant for bone calcification and remodelling are misregulated 301


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such as sost and ctsk. Thus, our study is the first to indicate the conservation of the proposed 302

pathway shown in cell culture in an in vivo model. 303

Weberian apparatus 304

The Weberian apparatus is a specialized structure that connects the auditory system and the 305

swim bladder in a complex with the first 4 vertebrae to amplify sound vibrations.(33) Our results 306

show that key structures in the apparatus fail to efficiently form and/or mineralize. Two of these 307

structures, the tripus and the parapophysis, are formed to connect to the swim bladder. Both are 308

malformed in all tested chd7-/- mutants to varying degrees. This malformation may imply an 309

inefficiency in its function concerning both the swim bladder and auditory system. Ear 310

abnormalities are a major symptom in CS patients and functionality is affected. Furthermore, 311

the chd7-/- mutant shows swim bladder defects (Breuer et al., unpublished) that could be 312

additionally affecting the morphology in tripus and parapophysis or vice versa. Even though 313

the Weberian apparatus is extremely specialized for carp and carp-like fish (Ostariophysi), it is 314

likely that it underlies the same pathways for osteogenesis as other parts of the spine. 315

Abnormal vertebrae and vBMD variability 316

We examined volumetric bone mineral density (vBMD) and other parameters (e.g. BV, TV) in 317

vertebral bone structures. Most strikingly, we detected large variance in density and all 318

parameters examined, as well as some significant changes in both precaudal and caudal 319

vertebrae. The large variance observed in chd7-/- mutants is in line with CS patients showing 320

varying severity in spinal deformities.(12, 59) Furthermore, previous studies have shown that 321

even minor changes in the described parameters can impact the risk for spinal deformities and 322

fractures in disease and under treatment.(60, 61) 323

Another interesting feature we observed in most chd7-/- mutants, were highly mineralized 324

inclusions, most commonly, localized towards the growth zone of the vertebrae. We did not 325


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identify the source of these highly mineralized structures, but they could be caused by highly 326

localized clusters of osteoblasts during vertebrae mineralization. Another possibility is the 327

occurrence of calcified cartilage, which is more mineralized in comparison to bone. Although 328

speculative, the occurrence of mineralized cartilage could be due to incomplete endochondral 329

ossification, which would be in line with the reduction in sp7/osterix and col2a1 expression(62, 330

63) Since these inclusions are highly mineralized, it seems likely that their appearance impairs 331

the bone structurally as higher mineralized collagen is stiffer, yet less tough.(64) It will be of 332

interest to see if future studies can take a closer look at these previously unidentified features. 333

While these minor defects could be involved in the structural integrity of the spine in CS 334

patients, these minor features may be missed during dual-energy x-ray absorptiometry (DEXA) 335

screens of CS patients when assessing aBMD. 336

Analysis revealed a decreased mineralization in the caudal vertebral arches of the mutant fish. 337

This likely reduces the mechanical competence of these vertebrae in comparison to healthy 338

vertebrae. A compromised mechanical competence alongside low BMD also increases fracture 339

risk and can be an underlying cause of scoliosis.(65-67) Unfortunately, natural history BMD data 340

of CS patients throughout early development is not available, to the best of our knowledge. 341

However, aBMD analysis of patients with idiopathic scoliosis in CS via DEXA scans has been 342

studied and shows significant reduction in some cases.(13, 68) In general, there is an increased 343

fracture risk and incidence of scoliosis corresponding to decreased BMD, such as in osteopenia 344

and osteoporosis.(47, 66) In line with this reduced mineralization an increased fracture and injury 345

risk has been reported for CS patients.(13) Accordingly, proper observation and focus on early 346

DEXA analysis is considered for CS guidelines.(45, 46) 347

Our analysis throughout development of chd7-/- deficient zebrafish suggests that only mild 348

phenotypes survive onto later stages with severity decreasing as age progresses. However, the 349

high lethality in both patients and our model is connected to a multisystemic phenotype in which 350


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one mechanism is unlikely to be exclusive underlying cause for this characteristic. The link 351

between Chd7 and severe spinal and vertebrae abnormalities, including kyphosis, fused 352

vertebrae and hemivertebrae has been observed in our samples.(7, 9) 353

Collagen and chondrocyte depletion as additional risk factor 354

Our data in chd7 deficient zebrafish indicate that reduced vBMD may not be the only 355

underlying risk factor for idiopathic scoliosis in CS patients. Spinal integrity may be weakened 356

by a decrease in chondrocyte marker col2a1 expression in the vertebral cartilage of chd7 357

deficient zebrafish. Col2a1 is required in the ECM of various cell types including osteoblasts, 358

chondrocytes, external ligament connective tissue cells, as well as notochord basal cells and 359

expressed in the corresponding tissues.(69, 70) Each cell type is required for proper integrity and 360

could likely contribute to spinal injuries and malformations. Furthermore, col2a1a expression 361

is observed throughout ossification of the notochord.(71) Reduction in this collagen matrix in 362

the notochord can affect proper calcification. Col2a1a is expressed in osteoblasts and 363

chondrocytes of teleosts and regulated by sox9, which in turn is known to be regulated by 364

Chd7.(72-74) The hypertrophic morphology of the chondrocytes seen in our study may indicate 365

signs of a weakened matrix, as well as altered matrix composition and may contribute to an 366

increased risk in osteoporosis.(75, 76) How this regulatory defect may influence other 367

cartilage/skeletal regions such as the craniofacial cartilage remains to be investigated. 368

In summary, our study is the first to identify skeletal abnormalities linked to chd7 deficiency. 369

These abnormalities include morphological changes in vertebrae, reduced bone mineralization, 370

impaired osteoblast activity, highly mineralized inclusions and Col2a1 deficiency in the chd7-371

/- zebrafish model organism. The use of this preclinical model will allow for future studies to 372

further understand the underlying mechanisms of spinal deformities in CS. 373

374


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Materials and Methods 375

Zebrafish husbandry 376

Wildtype (Cntrl) and mutant (chd7-/-) zebrafish were kept at 28°C in a 12h/12h dark/light cycle 377

and maintained in accordance with Westerfield et al.(77). All zebrafish in this study were fed a 378

steady diet of Skretting®GemmaMicro starting at 5dpf. Embryos were raised at 28.5°C and 379

staged as previously described by Kimmel et al.(78) All experiments were performed in with the 380

guidelines of the Canadian Council for Animal Care and the local ethics committee. 381

Skeletal stainings and analysis of larvae 382

Calcein staining was done as described(38). In short, zebrafish larvae at 9dpf were exposed to 383

calcein (2g/L) (Sigma-Aldrich) in water for 10 minutes. Larvae were then washed at least 3 384

times in fish water for 10 minutes to remove excess calcein. Larvae were then anesthetized 385

using tricaine before being imaged using an AxioZoom V16 (Zeiss). For nutrition dependency 386

tests, larvae were either deprived of food until 9 dpf or fed a regular Skretting®GemmaMicro 387

diet starting at 5 dpf. 388

Alizarin Red was performed as previously shown(79). In brief, larvae were fixed for 2 hours with 389

2% PFA. The PFA was then removed by washes in PBS 3x10 minutes and then placed in 25% 390

glycerol/0.1%KOH. Finally, larvae were stained with 0,05% Alizarin Red (Sigma-Aldrich) in 391

H2O for 30 minutes and stored in 50% glycerol in 0.1%KOH. 392

MicroCT Imaging 393

Adult zebrafish were euthanized using tricaine (MS-222, Sigma-Aldrich). Tissue was fixed in 394

4% PFA prior to microCT analysis. Ex vivo microCT at an isotropic voxel size of 10.6 µm 395

(SkyScan1276, Bruker, Kontich, Belgium) was performed to assess differences in bone mass, 396

mineral density, and microstructure. The following scanning parameters were used: peak 397

voltage of 55 kVp, Aluminum 0.25 mm filter, source current of 200 µA, 0.3° rotational steps 398


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for full 360° with no frame averaging. Images were reconstructed using standard reconstruction 399

algorithms provided with the microCT. Spinal deformities of controls and mutants were 400

analyzed of 1-year (n=8 for Cntrl and n=8 chd7-/-) and 2-year-old (n=5 for Cntrl and n=5 chd7-401

/-) zebrafish. 402

Analysis and bone density/volume/angles 403

From the reconstructed microCT images of the whole spine, the Weberian apparatus, as well as 404

vertebrae 7 (abdominal) and 20 (caudal) were segmented. Further, the intercalarium, the tripus 405

as well as the parapophysis were segmented from the Weberian apparatus. For the vertebrae, 406

the neural as well as the hemal arch were segmented from the vertebral body. To differentiate 407

between bone and background, a density-based threshold of 0.41 gHA/cm3 determined by 408

Otsu’s method(80) was used. For each segmented bone in the Weberian apparatus outcomes 409

included: bone volume BV (mm3), tissue volume TV (mm3), bone volume fraction BV/TV 410

(mm3/mm3) and volumetric bone mineral density vBMD (gHA/cm3). For abdominal and caudal 411

vertebrae the outcomes included: bone volume BV, tissue volume TV, bone volume fraction 412

BV/TV, total bone mineral density T.vBMD, vertebral body vBMD VB.BMD, vertebral arch 413

vBMD VA.BMD, as well as arch-body angle, body angle, arch opening angle and rising angles 414

(see Fig.2a and 3a). For caudal vertebrae, the angle outcomes were calculated for hemal as well 415

as neural arches. 416

qRT-PCR 417

Total RNA was isolated from 9 dpf old zebrafish larvae using TriReagent. 1µg of RNA was 418

used for cDNA synthesis using cDNA vilo kit. qRT-PCR was performed with SYBR Green 419

mix (BIORAD) with a Lightcycler96® (Roche). Gene expression was analyzed relative to the 420

housekeeping gene elf1α. Primers used for runx2a, runx2b, sost, bglap, sp7/osterix, mgp, 421

postna, acp5a, ctsk, col2a1 and mgp are shown in supplementals Table 5. 422


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Spinal sections/Stainings/Immunohistochemistry 423

The zebrafish were euthanized in tricaine (MS-222, Sigma-Aldrich) and fixed for 3 days in 4% 424

paraformaldehyde at 4°C, after the abdomen was opened to ensure proper fixation. The spinal 425

cords were dissected and decalcified with EDTA 10 % for 3 days under agitation at RT. After 426

fixation and decalcification, the zebrafish were embedded in paraffin. Longitudinal sections 427

(5μm) were obtained and were deparaffinized in xylene and were rehydrated in a graded series 428

of ethanol. The slides were stained with hematoxylin (STATLAB Medical Products, LLC) for 429

4 min, and washed with alcohol-acid, and were rinsed with tap water. In the blueing step, the 430

slides were soaked in saturated lithium carbonate solution for 10 sec, and then rinsed with tap 431

water. Finally, staining was performed with eosin Y (STATLAB Medical Products, LLC) for 2 432

min and mounted with Permount™ mounting medium. 433

For Safranin O/Fast green, after dehydration, the slides were stained with Weigert’s 434

hematoxylin (Sigma-Aldrich) for 4 min, wash under tap water, immersed in alcohol–acid for 5 435

sec, then washed under water. The slides were stained with Fast green 0.02 % for 2 min, washed 436

with acetic water 1% for 20 sec, then stained directly in safranin 0.01 % for 5 min and mounted 437

with Permount™ mounting medium. Images were taken using an AxioZoom V16 (Zeiss) and 438

area and number of nuclei were determined using ImageJ. 439

Immunofluorescence for Col2a1 was performed as previously shown69. In short, Epitope 440

demasking was performed by digestion with Proteinase K (20µg/ml). Tissue was blocked with 441

NGS and samples treated with primary antibody for Col2a1 (1 in 10; Developmental studies 442

hybridoma bank) and secondary antibody Goat-anti-Mouse Alexafluor 488 (1 in 300). Counter 443

stain was done using DAPI-mounting medium. Images were taken using a LSM780 (Zeiss). 444

Statistics 445


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Statistical analysis was carried out using Prism-GraphPad® (GraphPad, San Diego, CA, USA). 446

qPCR data and nuclei quantification were tested with student’s t-test. Calcification analysis in 447

larvae was performed with one-way ANOVA and post-hoc Tukey Test. MiroCT data of Control 448

vs chd7-/- mutants was analyzed using student’s t-test with Welch’s correction and Variance 449

determined by F-test analysis. Significance was determined at *<0,05; **<0,01 and ***<0,001 450

Acknowledgements 451

The authors would like to thank Beatrice Steyn for experimental support and Mark Lepik for 452

graphic design. SP is supported by the Natural Science and Engineering Research Council 453

(NSERC), Canadian Institutes of Health Research (CIHR), an ALS Canada-Brain Canada 454

Career Transition Award and a FRQS Junior 1 research scholar. BMW is supported by FRQS 455

Programme de bourses de chercheur. MR is supported by FRQS Programme de bourses de 456

chercheur. We also thank Shriners Hospital for Children and the CIHR (165939) for financial 457

support. 458

Author roles 459

MB, MR, BMW and SP designed the study. MB, MR and CZ performed the experiments. MB 460

and MR drafted the manuscript. MB, MR, CZ, BMW, SP edited the mansucript. 461

462

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71. Dale RM, Topczewski J. Identification of an evolutionarily conserved regulatory 640 element of the zebrafish col2a1a gene. Dev Biol. 2011;357(2):518-31. 641 72. Akiyama H, Chaboissier MC, Martin JF, Schedl A, de Crombrugghe B. The transcription factor 642 Sox9 has essential roles in successive steps of the chondrocyte differentiation pathway and is required 643 for expression of Sox5 and Sox6. Genes Dev. 2002;16(21):2813-28. 644 73. Bajpai R, Chen DA, Rada-Iglesias A, Zhang J, Xiong Y, Helms J, et al. CHD7 cooperates with PBAF 645 to control multipotent neural crest formation. Nature. 2010;463(7283):958-62. 646 74. Eames BF, Amores A, Yan YL, Postlethwait JH. Evolution of the osteoblast: skeletogenesis in gar 647 and zebrafish. BMC Evol Biol. 2012;12:27. 648 75. Hall AC. The Role of Chondrocyte Morphology and Volume in Controlling Phenotype-649 Implications for Osteoarthritis, Cartilage Repair, and Cartilage Engineering. Curr Rheumatol Rep. 650 2019;21(8):38. 651 76. Lian C, Wang X, Qiu X, Wu Z, Gao B, Liu L, et al. Collagen type II suppresses articular chondrocyte 652 hypertrophy and osteoarthritis progression by promoting integrin beta1-SMAD1 interaction. Bone Res. 653 2019;7:8. 654 77. Westerfield M. The zebrafish book : a guide for the laboratory use of zebrafish (Danio rerio) 655 4th ed. [Text.]. Eugene, OR: Institute of Neuroscience, University of Oregon ,; 2000 [Available from: 656 http://zfish.uoregon.edu/zf_info/zfbook/zfbk.html. 657 78. Kimmel CB, Ballard WW, Kimmel SR, Ullmann B, Schilling TF. Stages of embryonic development 658 of the zebrafish. Dev Dyn. 1995;203(3):253-310. 659 79. Aceto J, Nourizadeh-Lillabadi R, Maree R, Dardenne N, Jeanray N, Wehenkel L, et al. Zebrafish 660 Bone and General Physiology Are Differently Affected by Hormones or Changes in Gravity. PLoS One. 661 2015;10(6):e0126928. 662 80. Otsu N. A Threshold Selection Method from Gray-Level Histograms. IEEE Transactions on 663 Systems, Man, and Cybernetics. 1979;9(1):62-6. 664

665


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666

Fig 1. Mineralization in larval and juvenile chd7-/- mutants 667

a. Dorsal view of control and chd7-/- larvae at 9dpf showing severe spinal deformities in 43 out of 110 668

larvae screened. Deformities were present at both precaudal and caudal region (arrows). b. Lateral view 669

of calcein staining at 9dpf showing mineralization of vertebrae with access to food starting at 5dpf and 670

c. calcein staining at 9dpf during starvation. Arrow indicates mineralization at the first 4 (Weberian) 671

vertebrae d. Number of vertebrae in wildtype and chd7-/- (fed and starved) larvae showing at least partial 672

calcification signal as tested by calcein and e. Number of weberian vertebrae (vertebrae 1-4) showing 673

complete calcification (see arrows in b) f. Lateral view of 4 week old zebrafish stained with alizarin red 674

to screen mineralized vertebrae, showing decreased calcification towards posterior region. Bottom right 675

corners show magnification of selected caudal vertebrae. g. RT-qPCR of osteogenesis related genes 676

screened at 9dpf, showing relative fold change. (Significance: *p<0.05; **p<0.01) 677


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678

Fig 2. Weberian apparatus 679

a. Representative microCT overview image of the spinal cord from a 1-year old control and a chd7-/- 680

adult zebrafish b. MicroCT Image of the Weberian apparatus from a 1-year old control and a chd7-/- 681

mutants, indicating structures of intercalarium (red), tripus (yellow) and parapophysis (blue). c-h. 682

vBMD and BV/TV of intercalarium (c, d) tripus (e, f) and parapophysis (g, h) (n=8/genotype were 683

analyzed). Significance of student t-test with Welch’s correction is included in the graphs with: *p<0.05; 684

**p<0.01. F-test results are indicated underneath as Fdegree of freedom numerator, degree of freedom denominator (F-value); 685

p-value: *p<0.05; **p<0.01; ***p<0.001 686


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687

Fig 3. Precaudal vertebrae show inefficiency for proper mineralization 688

a. Sketch diagram of precaudal vertebrae (frontal and lateral) indicating structure and measured angles. 689

b-c. (top) Individual rendering of precaudal vertebrae, frontal view of tissue over 0.41 g HA/cm3 690

threshold and (bottom) vBMD intensity map (Range blue-red; 0.41 – 1.00 g HA/cm3) showing 691

morphological abnormalities and growth zone malformations with highly mineralized inclusions 692

(arrow) d. vBMD of whole vertebrae showing increasing density with increasing age and e. increasing 693

ratio of vBMD arch/vertebrae body. f-h neural arch angle, BV, TV and ratio BV/TV. (n=8/genotype 694

were analyzed). Significance of student t-test with Welch’s correction is included in the graphs with: 695

*p<0.05; **p<0.01. F-test results are indicated underneath as Fdegree of freedom numerator, degree of freedom denominator 696

(F-value); p-value: *p<0.05; **p<0.01; ***p<0.001 697


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698

Fig 4. Caudal vertebrae show abnormal structure 699

a. Sketch diagram of precaudal vertebrae indicating structure and measured angles (frontal and lateral). 700

b-c. (top) Individual rendering of caudal vertebrae, frontal view of tissue over 0.41 g HA/cm3 threshold 701

and (bottom) vBMD intensity map (Range blue-red; 0.41 – 1.00 g HA/cm3) showing morphological 702

abnormalities and growth zone malformations with highly mineralized inclusions (arrow) d. vBMD of 703

whole vertebrae and E. increasing ratio of vBMD arch/vertebrae body. f-h neural arch angle, BV, TV 704

and ratio BV/TV. (n=8/genotype were analyzed). Significance of student t-test with Welch’s correction 705

is included in the graphs with: *p<0.05; **p<0.01. F-test results are indicated underneath as Fdegree of 706

freedom numerator, degree of freedom denominator (F-value); p-value: *p<0.05; **p<0.01; ***p<0.001 707


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708

Fig 5. ECM Collagen deficiency in 1-year-old chd7-/- adults 709

a. H&E staining of precaudal vertebrae section of Cntrl and chd7-/- mutants b. Total nuclei count in 710

precaudal vertebral cartilage detected in H&E staining per 100μm2 c. Safranin O/fast green staining of 711

precaudal vertebrae section Cntrl and chd7-/- mutants showing cartilage in red. d. Chondrocyte nuclei 712

count in precaudal vertebral cartilage detected in Safranin O/fast green staining per 100μm2 e. qPCR of 713

col2a1a at 9dpf. f. Immunofluorescence of Col2a1 in precaudal vertebrae sections of Cntrl and chd7-/- 714

mutants with Col2a1 in green and DAPI in blue. Scale bar presents 100μm. Significance of student t-715

test with: *p<0.05; **p<0.01 ***p<0.001 716


https://doi.org/10.1101/2020.07.10.197533

Altered vertebrae morphology and bone mineralization in a ...Jul 10, 2020 · 3 56 Introduction 57...

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